Caloric restriction (CR) is the surest way of increasing life span and delaying the onset of age-related symptoms in animals. During the current funding period, we made significant contributions to our understanding of the effects of age and caloric intake on physiology and longevity of adult Drosophila. Mutation in the Indy gene in the fruit fly, Drosophila melanogaster, dramatically extends life span. INDY is a dicarboxylate transporter of Krebs cycle intermediates primarily found in the tissues important for intermediary metabolism. The life extending effect of reduced Indy activity has been proposed to result in a form of genetic CR, a hypothesis supported by biochemical, molecular and genetic studies carried out during this funding period. Determination of the genomic transcriptional responses of Indy long-lived flies reveal down-regulation of genes that function in metabolism-particularly noteworthy is a transient decrease in the expression of components of the mitochondrial oxidative phosphorylation (OP) complexes I and III. We showed that in Indy flies OP I and III complex have lower enzyme activity, produced less reactive oxygen species (ROS), and caused lower oxidative damage. However, production of ATP in Indy flies is similar to the control, a result that could be explained by increased mitochondrial density found in Indy flies. Considering the crucial role of mitochondria in energy production and cellular homeostasis, our preliminary data provide additional links between metabolic and longevity pathways and form the basis for our hypothesis that transient change in the OP complexes mediate longevity in Indy mutant flies. In parallel, we have shown that down-regulation of the rpd3 histone deacetylase, or overexpression of dSir2 histone deacetylase genetically, or increasing dSir2 activity by feeding flies resveratrol, extends life span in Drosophila by a mechanism similar to CR. This give us an opportunity to determine if similar changes in mitochondrial physiology are part of the pathway underlying life span extension in three fly models of genetic CR. In aim 1 of this proposal, we will determine if genetic manipulations of Indy, rpd3, and Sir2 genes, or CR, effect longevity by downregulation of the levels and activity of the OP I and III components in each life span extending condition. In aim 2, we will determine if decreasing the levels of components of complex I and III have effects on fly physiology and longevity. In aim 3, we will examine genetic interactions between OP I and III components and the established Indy/rpd3/Sir2 longevity pathway. In aim 4, we will further elucidate the role mitochondria play in CR life span extension by assessing the mitochondrial physiology and biogenesis in the Indy/rpd3/Sir2 longevity pathway. Since the role of mitochondria in energy homeostasis, stress response and longevity is well known, our proposed experiments will extend current knowledge to the novel role of the OP components in the CR pathway and potentially provide a basis for therapeutic intervention. PUBLIC HEALTH RELEVANCE: Caloric restriction has emerged as the most efficient way to protect the organism against deleterious effects of aging in both vertebrate and invertebrate species. This project will study the molecular mechanism underlying life span extension in fruit flies, Drosophila melanogaster, by caloric restriction. It will reveal how reduced caloric intake affects metabolism and life span, and provide the foundation for the development of new therapies for the treatments of age-associated diseases in humans.